1. Technical Field of the Invention
The invention relates to a process for the quantitative determination of at least one parameter of a liquid or gaseous sample, in which process the fluorescence radiation of an indicator substance in direct or diffusion contact with the sample is measured, said fluorescence radiation being emitted following fluorescence excitation.
2. Discussion of the Related Art
In known measuring devices with optically active indicator substances or fluorescent dyes, the change in the excitation or emission spectrum that occurs with a change in the parameters to be measured or in the concentration of the substance to be determined is recorded, in which process the desired parameters or concentrations of the substance can be determined by means of suitable calibrating processes and calibrating substances. The terms "excitation spectrum" and "emission spectrum" shall be defined here and in the following manner, to obtain the excitation spectrum the wavelength of the excitation radiation is varied over its spectrum and the intensity of the emission radiation is measured at a specific wavelength. To obtain the emission spectrum the wavelength of the excitation radiation is held constant and the spectral intensity distribution of the emission radiation is measured. The excitation radiation is monochromatized in the conventional manner by means of suitable devices such as selective filters or prisms with subsequent slit diaphragm or with the aid of electronically tunable excitation light sources. The emission radiation is recorded, also as a function of the wavelengths, by means of a suitable measuring device.
Thus, for example, a measuring device, which has an indicator chamber that on the sample side is covered with a selectively permeable membrane and in which there is an organic indicator substance, is known from the DE-08 2 508 637. In this known system specific organic compounds such as .beta.-methyl umbelliferon are used as indicators that react with a change in their excitation spectrum in the solution containing the indicator, independent of the concentration of positive hydrogen ions. Thus in enabling the selective diffusion of hydrogen ions from the sample to be measured into the indicator chamber or the indicator solution, the hydrogen ion concentration or the pH value of the sample can also be measured.
It is also known from this document that the intensity of the emission spectrum of specific organic compounds such as the pyrene butyric acid is reduced by means of the presence of molecular oxygen on a scale that is largely comparable to the partial pressure of oxygen in the phase containing the indicator, with which such a device can also be used to measure the partial pressure of oxygen.
However, this known system has the drawback that the simultaneous measurement of the concentration of several different substances in the sample is relatively expensive. In addition to this, several indicator chambers lying next to one another are mandatory, each of which must contain an indicator substance that responds to the substance to be measured and which is excited via the respectively assigned monochromators and whose fluorescence radiation is evaluated by means of the corresponding suitable measuring devices.
Furthermore, it is known from AT-PS 377 364 that a substituted aromatic or heteroaromatic compound can be used as the organic indicator substance, in which process the substituted aromatic or heteroaromatic compound contains one or more substituents. Such indicators or indicator substances, which react in a clearly distinguishable manner to the change in the concentration of at least two different substances in the sample by a shade change in the excitation spectrum and intensity change in the emission spectrum, the change in the shade and the change in the intensity serving as a measure for the change in concentration of the substances to be detected, have as functional groups, in particular, at least one phenol, carboxylic acid, or sulfonic acid group, etc. or a combination of these groups. Thus organic indicator substances are used here that react to the change in the concentration of the substances to be measured in the sample by a shade alteration or intensity change in the fluorescence radiation emitted following excitation.
For example, an indicator substance can be used that reacts with a shade alteration of its excitation spectrum when the concentration of hydrogen ions in the sample changes and with an intensity change in its emission spectrum when the concentration of molecular oxygen in the sample changes, whereby the isosbestic wavelength remains at least approximately constant. When excited with the isosbestic wavelength, the intensity of the fluorescent light is by definition a pH independent variable and represents a measure for the entire concentration of the indicator substance contained in the indicator chamber.
There are certain drawbacks with this method since at least one sought parameter (here O.sub.2) is a function exclusively of an intensity change in the fluorescence radiation and thus is a function of the aging processes (photo bleaching) of the indicator substance. In addition to this, fluctuations in the intensity of the excitation light source and the varying response sensitivity of the measuring device detecting the fluorescence radiation influence this measured value.
Since the definitive separation of the weakening and the shade alteration of the fluorescence radiation and the respective assignment to a change in the hydrogen ion concentration or change in the concentration of molecular oxygen in the sample is possible only if the isosbestic wavelength remains at least approximately constant at different O.sub.2 concentrations, a selection criterion is assumed that is fulfilled only by few indicator substances.
The object of the present invention is to avoid the drawbacks of the indicator substances used for the aforementioned devices and/or processes and, thus, to improve in a simple manner the simultaneous measurement of two parameters of a sample, in which process the measured values shall not be influenced by the aging processes of the indicator substance, fluctuations in the light intensity of the excitation light source, and different detector efficiency.